Moynihan Patrick J, Clarke Anthony J
From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
J Biol Chem. 2014 Jun 13;289(24):16748-60. doi: 10.1074/jbc.M114.567388. Epub 2014 May 2.
The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.
必需的细胞壁聚合物肽聚糖的O-乙酰化是在许多细菌中鉴定出的主要毒力因子，包括革兰氏阳性菌和革兰氏阴性菌，如金黄色葡萄球菌、炭疽芽孢杆菌、淋病奈瑟菌和脑膜炎奈瑟菌。对于革兰氏阴性菌，乙酰基团从细胞质的转运由整合膜蛋白PatA执行，以便通过O-乙酰转移酶PatB将其转移到肽聚糖上，而单一的双峰膜蛋白OatA似乎在革兰氏阳性菌中催化该过程的两个反应。此前仅有支持这些途径的表型证据，因为没有体外生化分析方法可用于分析它们，这反映了研究作用于完全不溶性和异质底物（如肽聚糖）的整合膜蛋白的复杂性。在本研究中，我们以淋病奈瑟菌的PatB为模型系统，首次对肽聚糖O-乙酰转移酶进行了生化和动力学分析。该酶对在胞壁酰残基上具有三肽和四肽茎的胞壁肽具有特异性。以壳寡糖为底物时，反应速率随着聚合度增加至5/6而增加。这些信息对于鉴定和开发肽聚糖O-乙酰转移酶抑制剂具有重要价值，这些抑制剂可能成为新型抗生素类别的潜在先导物。
